Structure and interactions of the Clostridium difficile S-layer with bacteriocins.

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Clostridium difficile is an important human pathogen, causing serious illness and even death. C. difficile infection (CDI) occurs most commonly in the hospital setting, affecting patients who are already suffering ill health, but infections in the community are an increasing problem. C. difficile is naturally resistant to many common antibiotics so whilst these antibiotics kill the beneficial bacteria in the human gut they have no effect on C. difficile; indeed they actually benefit the bacterium by removing competition. The proteins on the surface of the C. difficile bacterium are the appendages through which it interacts with its environment and the human host. The C. difficile surface is coated in a single layer of protein that forms a two-dimensional crystal that wraps around the bacterium. This surface layer (S-layer) acts as a coat of armour to protect the bacterium from attack by our immune system and is essential for the bacteria to cause disease. Despite the importance of the S-layer we know very little about how it assembles on the cell surface and what it looks like. We have recently collaborated with a biotech company who have developed new therapeutic particles that attach to the S-layer to kill the C. difficile. These particles, called Avidocins, consist of a needle inside a spring-loaded sheath. When the Avidocin binds to the cell surface the sheath contracts and drives the needle through the cell wall and membrane, killing the bacterium. Although we know that Avidocins bind to the S-layer we do not know exactly how they recognise the S-layer, where they bind, what changes they undergo while binding, how the contraction is triggered or how the needle penetrates the various layers of the cell envelope.In this project we will combine our complementary expertise in C. difficile biology and powerful electron microscopy to:1. Understand the structure and organisation of the S-layer. This is essential information if we want to develop new treatments that target the S-layer to treat or prevent CDI.2. Understand the structure of the Avidocin particle both on its own and in the act of binding to and killing a C. difficile cell.There is an urgent need to develop new therapies to combat CDI and, in particular, to develop therapies which kill C. difficile but do not cause damage to the beneficial gut bacteria. A new therapy would ideally target a distinctive part of the bacterium which is essential for its lifecycle. The S-layer is an ideal candidate for this sort of intervention because it doesn't resemble the surface of any other kind of bacteria. Avidocins are promising therapeutic agents in their own right and are also closely related to natural viruses that infect C. difficile (bacteriophage). There is a lot of interest in the potential of bacteriophage to treat infections. Many of the features of Avidocin killing that we will study are directly relevant to bacteriophage infection, including binding to the S-layer, contraction of the spring-loaded sheath and penetration of the cell envelope. Our work on S-layer will identify ways in which this important structure can be targeted to tackle CDI.